In many research and analysis fields, the objective is to determine the presence or absence of some particles, so-called “particles of interest”, in a liquid and/or to study the development of these particles of interest in this liquid and/or to promote the formation of structures from the particles in the liquid. These particles may be, on the one hand, biological objects such as cells (animal and/or vegetal, living and/or non-living cells), antibodies, proteins, viruses, etc. . . . and/or, on the other hand, non-biological objects such as molecules, so-called molecules of interest, including chemical molecules in particular polymers, liquids, etc.
Most of the protocols, implemented for the analysis and/or the study of a sample generally consisting of a liquid containing (or not) at least one particle, use a sample support which is frequently constituted of a parallelepipedic glass slide and an observation instrument such as a microscope, a spectrum analyzer, a fluorescence quantization system, etc. The sample is placed onto the glass slide, then a cover slip is gently placed onto the sample as illustrated in FIGS. 3A to 3C in the present specification, so as not to enclose air bubbles between the lower surface of the cover slip and the sample nor to damage the particles possibly present in the sample, the cover slip being then maintained by capillarity on the glass slide. In some applications, the cover slip can be fixed to the glass slide by adhesive bonding, generally with the help of wax, in particular in order to enclose the sample and avoid the evaporation thereof which may occur during continuous observations especially of long duration and/or when the sample is subjected to temperature variations, in particular temperature rises.
When limiting the evaporation of the liquid, the adhesive bonding technique makes it possible, on the one hand, to avoid or limit the movement of the liquid in the analysis zone of the sample and, on the other hand, to avoid a variation in the concentration of particles and/or salts possibly dissolved in the sample, which allows an observation of the particles under stabilized conditions.
However, the use of this type of protocols causes many difficulties for the user. First, when the liquid is deposited onto the slide, the drop can spread in an uncontrolled manner on the surface into a film a few microns thick. In addition to the sample loss along the edges of the blade, it then becomes difficult to control the lateral extension of the liquid, for example relative to an analysis zone imposed by the analysis instrument.
Other capillary phenomena can also disturb the positioning of the sample: when the cover slip is placed, it is common that the affinity of the liquid for one of the two substrates causes the drop to quickly migrate to one of the edges of the slide and the cover slip. This phenomenon is also at the origin of the wasting of a part of the sample and makes the positioning of the sample in the center of the cover slip more complex. The sample then gets a random lateral extension, and therefore has a thickness difficult to control. Finally, the presence of liquid at the edge of the cover slip can disrupt the adhesive bonding step which makes it possible to make the system hermetic. Since this step depends on the dexterity of the user, the result of conditioning the sample in the form of a thin cavity is very random and this can result in a large variability of the analysis results. The presence of liquid outside the slide and/or cover slip, which can thus be in contact with the user and/or the analysis instrument, is generally considered unacceptable particularly in the case of samples containing carcinogenic or toxic products.
Finally, the adhesive bonding step take a particularly long time to get implemented and requires the intervention of an experienced user. For this reason, this step is not possible for certain applications that require the analysis of a large number of samples. In addition, it does not allow the user to recover her/his sample since this type of adhesive bonding is not suitable for a reversible opening. In an attempt to find a remedy for some of these disadvantages, various solutions have already been proposed.
U.S. Pat. No. 2,041,290A discloses a sample holder in the form of a plate for the analysis of a liquid sample, for example a drop of blood, on the upper surface of which a plurality of circular wells is provided, each well being surrounded by a channel for collecting the excess of liquid from the sample to be analyzed in said well.
U.S. Pat. No. 2,302,830A discloses a plate of the same type in which the well is formed by depositing a circular bead of material onto the surface of the slide, this bead being surrounded by a circular groove etched in the surface of the slide so as to collect the excess of liquid to be analyzed.
U.S. Pat. No. 5,948,685A proposes to use a device consisting of a microscope glass slide on which a retention barrier less than 1 μm thick is structured for providing a well in the surface of the slide. As the sample is placed inside this well, any overflow is in principle stopped, which makes it possible to avoid the effects of loss of liquid at the edges of the cover slip. However, it has been found that, if the volume of the sample is poorly controlled, i.e. it is too big, this barrier is not sufficient to prevent it from leaking at the edges. In addition, if the cover slip that is just placed on the sample has a high affinity with the liquid sample, which is often the case and if it is not deposited strictly parallel to the slide, a liquid overflow event takes place along this cover slip.
WO 82/02958 discloses a support plate for the analysis of liquid samples, this pate comprising a plurality of assemblies for depositing a drop of liquid sample, each assembly being composed of a central well surrounded by a channel or groove capable of receiving the excess liquid deposited in the central well.
U.S. Pat. No. 7,138,270B2 describes in particular in FIG. 8 a support for liquid samples comprising a plurality of circular wells regularly distributed on the upper surface of the support. The typical height of the side walls of the wells is between 30 and 200 μm, all of these wells being surrounded by an external retention barrier. This type of configuration makes it possible to completely fill the wells with the help of an excess of liquid which is removed during the placement of the upper cover slip, in the intermediate zone situated between the different wells and delimited, on the one hand, by the lateral walls of the various wells and, on the other hand, by the external retention barrier. This external barrier makes it possible to retain the excess liquid to avoid any leakage outside the slide and the cover slip. In this type of system, the positioning of the excess liquid is however not controlled and it is necessary to deposit the cover slip in a suitable manner so as to limit cross contamination between the wells. Indeed, as long as the upper plate is not hermetically sealed to the structures of the wells, some of the excess liquid can be drained by capillarity out of a well in an uncontrolled manner. This has the effect of generating a bubble within one or more wells (if the amount of liquid drained is too great) and the diffusion of the particles from one well to another when the liquids overflowing from two consecutive wells mix to one another, thus contaminating the subsequent analysis of initially independent wells. In addition, the slightest dust or particle from the sample coming between the barrier and the upper cover slip or the slightest irregularity in the barrier is at the origin of a preferred non-controllable evaporation zone which limits the reproducibility of the analysis, especially when the latter takes time and requires to be performed at high temperatures. This configuration is therefore unreliable for routine analysis even if it is performed with particularly reproducible (and therefore expensive) technologies.
In order to limit the problems of evaporation and leakage, it has been proposed in WO 2006/052492A1 to first deposit a layer of rough glue on the retention barrier. The disadvantage of this solution is the impossibility to recover the sample, the loss of performance of this type of device in case of dust or defects in the manufacturing process and especially the risk of diffusion of the glue compounds in the sample to be analyzed which would then be contaminated.
US 2003/0194709A1 describes a method for producing a substrate comprising electrodes of hydrophilic material partially surrounded by hydrophobic zones in order to perform an electrochemical analysis of a DNA-type sample. The use of such a substrate does not require the use of a protective cover slip deposited on the liquid sample, this cover slip being used only for carrying out optical analyzes of a sample. Thus, the problem associated with the presence of an air bubble cannot exist with this type of substrate which is used without any cover slip.
None of the above-mentioned systems therefore makes it possible to provide a satisfactory solution to the problem of positioning and maintaining a liquid sample in a fine cavity, which allows precise lateral positioning of the sample, an excellent fluidic and chemical stability of the sample in the measurement area, as well as an absence of cross-contamination in the case of positioning multiple samples on the support, regardless of the user's dexterity and the accuracy of the sample volume.
Furthermore, when the supports such as described above are used with a cover slip (usually made of transparent glass or any other similar material) which comes to rest on the liquid sample in order to trap it between the cavity and the cover slip, so as to obtain an observable sample directly above the cover slip, it is very common to note that a gas bubble was formed in the liquid at the cavity, under the cover slip. The presence of this bubble that can move throughout the cavity makes the observation of the liquid sample difficult if not impossible, especially when the surface occupied by the bubble is large relative to the surface of the cavity. In an attempt to avoid the formation of this bubble due to a lack of liquid in the cavity, the experimenter tends to provide a substantially greater quantity of liquid relative to the volume of the cavity. However, it was found that this did not prevent the formation of this bubble when placing the cover slip onto a support such as described above.